D.Nirupama et.

al / IJIPSR / 2 (7), 2014, 1428-1437

ISSN (online) 2347-2154

RESEARCH ARTICLE
Department of Pharmaceutical Analysis

International Journal of Innovative

Pharmaceutical Sciences and Research
www.ijipsr.com
METHOD DEVELOPMENT AND VALIDATION FOR
METFORMIN HYDROCHLORIDE AND GLICLAZIDE IN
BULK AND PHARMACEUTICAL FORMULATION BY RPHPLC METHOD
1

D.Nirupama *, 2P.Venkateswara Rao, 3B.Thangabalan, 4 S.Manohar Babu

Department of Pharmaceutical Analysis, Sims College of Pharmacy, Mangaldas Nagar, Opp

Best Prize Vijayawada road, Guntur, INDIA
Abstract
A RP-HPLC method was developed and validated for the simultaneous estimation of Metformin
Hydrochloride (MET) and Gliclazide (GCL) in pure and pharmaceutical dosage form. Chromatography
was carried on Phenomex (kromosil-250 mm 4.6 mm, 5 m) column with mobile phase comprising of
phosphate buffer and methanol in the ratio 50:50 v/v. The flow rate was adjusted to 1.0 ml/min with UV
detection at230 nm. The retention times of MET and GCL were found to be 3.2 min, 5.0 min respectively.
The different analytical parameters such as accuracy, linearity, precision, robustness, limit of detection
(LOD), limit of quantification (LOQ) were determined according to the ICH-Q2B guidelines. The detector
response was linear in the range of 62.5-625 g/ml, 10-100 g/ml for MET, GCL respectively. The
proposed RP-HPLC method is sensitive, precise and accurate so it was successfully applied for the reliable
quantification of drugs in the commercial dosage form.

Key words: Metformin hydrochloride, Gliclazide, RP-HPLC and Simultaneous estimation.

Corresponding author
D.Nirupama
Department of Pharmaceutical Analysis,
Sims College of Pharmacy, Mangaldas Nagar,
Opp Bestprize, Guntur, INDIA
Email: dnreddy.niru@gmail.com
Phone: +91 9603210830
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RESEARCH ARTICLE
Department of Pharmaceutical Analysis

INTRODUCTION
Gliclazide 3-[(3aR,6aS)-octahydrocyclopenta[c]pyrrol-2-yl]-1-(4-methylbenzenesulfonyl)urea is
an oral hypoglycaemic agent used in treatment of diabetes mellitus type II [1,2]. Different
analytical methods including radioimmunoassay [3,4] gas chromatography [5], HPLC ,
Evaporative Light Scattering Detection, Charged Aerosol Detection

and simultaneous

spectrophotometric estimation of gliclazide and metformin hydrochloride in combined dosage

forms have been reported for determination of gliclazide. Metformin Hydrochloride is N, N-d
imethylimido di carbonimidic diamide hydrochloride chemically. Metformin activates AMPactivated protein kinase (AMPK), a liver enzyme that plays an important role in insulin
signaling, whole body energy balance, and the metabolism of glucose and fats; activation of
AMPK is required for metformin's inhibitory effect on the production of glucose by liver cells
[6,7]. In addition to suppressing hepatic glucose production, metformin increases insulin
sensitivity, enhances peripheral glucose uptake, increases fatty acid oxidation, and decreases
absorption of glucose from the gastrointestinal tract. Increased peripheral utilization of glucose
may be due to improved insulin binding to insulin receptors [8]. AMPK probably also plays a
role, as Metformin administration increases AMPK activity in skeletal muscle.

MATERIAL & METHODS

Table 1: Instruments\equipments\Apparatus used for the method development
S.No

Instruments/Equipments/Apparatus

High performance liquid chromatography (Waters e 2487)

Electronic Balance (Shimadzu)

Ultra Sonicator (Fast-Clean)

Analytical Balance (Shimadzu)

A Waters symmetry shield Phenomex (kromosil-250 mm 4.6 mm, 5 m)

Chemicals used
Metformin (standard), Gliclazide (standard), Methanol (AR grade), Phosphate buffer, Water(
HPLC grade)

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RESEARCH ARTICLE
Department of Pharmaceutical Analysis

METHOD DEVELOPMENT
Reference standards
Gliclazide and Metformin
Strengths of dosage forms
Brand name: Aviglic -MF
Preparation of mobile phase
Accurately weighed portion of 2.722g of potassium dihydrogen orthophosphate was dissolved in
200 ml of HPLC water. Separately 700mg of di-sodium hydrogen orthophosphate was weighed
and dissolved in 20ml of HPLC water, the pH adjusted to 6.5 using di sodium hydrogen ortho
phosphate, then the solution was filtered through a 0.22m filter membrane and stored in closed
container.
Standard stock solution preparation
Accurately weighed 62.5mg of Metformin and 10mg of gliclazide was dissolved in diuent, in
100 ml volumetric flask, that gave 625 g/ml of Metformin and 100 g/ml of Gliclazide. From
this into a series of five 10 ml volumetric flasks 1, 2, 4, 6, 8, 10ml were transferred and diluted to
10 ml with diluent, that gave 10, 20, 40, 60, 80, and 100 g/ml of Gliclazide and 62.5, 125, 250,
375, 500 and 625 g/ml of Metformin.
Sample stock solution preparation
. 20 tablets of combined formulation of Metformin and Gliclazide were weighed, average weight
was calculated and triturated in a mortar with pestle from that, powder equivalent to 62.5 mg of
Metformin and 10 mg of Gliclazide was weighed and dissolved in diluent and test concentration
was prepared by further dilution with same.
Table 2: Optimized Chromatographic conditions

Stationary phase (column)

A Waters symmetry shield

Rp18,(250x4.6x5 column

Mobile phase

Phosphate buffer:methanol. ( 50:50 )

pH

6.5

Flow rate (ml/min)

1ml/min

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RESEARCH ARTICLE
Department of Pharmaceutical Analysis

Run time (minutes)

7 mins

Column temperature (0C)

300C

Volume of injection loop (l)

10

Detection wavelength(nm)

230nm

Drug

Gliclazide and Metformin

Drug Rt (min)

5.001 & 3.232

Method precision:
Table 3: Results of Metformin for precision studies
S. NO

RETENTION TIME

PEAK AREA

ASSAY %

5.005

3503557

99.72

5.007

3505877

99.79

3
4
5
6

5.006
5.006
5.006
5.004
%RSD

3503969
3508933
3501948
3501546
--

99.73
99.87
99.67
99.66
0.08

Table 4: Results of Gliclazide for precision studies

S.NO

RETENTION TIME

PEAK AREA

ASSAY %

3.234

3152466

99.10

2
3
4
5
6

3.233
3.231
3.231
3.229
3.230

3151745
3158954
3157787
3158663
3155433

99.08
99.31
99.27
99.30
99.19

--

0.10

%RSD

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RESEARCH ARTICLE
Department of Pharmaceutical Analysis

ACCURACY
Table 5: Results for Accuracy of Metformin
Conc.
125
125
125
125
125
125
250
250
250
375
375
375
375
375
375

Retention time
5.009
5.009
5.003
5.003
4.995
4.991

Peak area
1751636
1751270
1758366
1756333
1750913
1750708

%recovery
99.87
99.85
100
100
99.83

4.982
4.978
4.977

3508264
3509457
3506370

100
100

4.971
4.972
4.971
4.974
4.973
4.968

5253135
5250130
5251467
5259637
5255756
5253042

Mean recovery

99.89

99.81
99.98

99.95
99.59
99.53
99.56
99.71
99.64

99.6

99.59

Table 6: Results for Accuracy of Gliclazide

Conc.
20
20
20
20
20
20

Retention time
3.236
3.236
3.231
3.233
3.232
3.230

Peak area
1579162
1576213
1573695
1570450
1576050
1573005

%recovery
100
99.85
99.69
99.49
99.84
99.65

40
40
40

3.222
3.220
3.220

3152275
3151260
3157232

99.85
99.82
100

99.89

60
60
60
60
60
60

3.216
3.216
3.216
3.216
3.217
3.210

4726940
4729848
4723140
4728536
4728895
4724036

99.58
99.64
99.50
99.61
99.62
99.51

99.58

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Mean recovery

99.75

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RESEARCH ARTICLE
Department of Pharmaceutical Analysis

LINEARITY:
Table 7: Linearity Studies of Metformin
S.No

Sampel Name

Inj

Name

RT

Area

LINEARITY-62.5%

Metformin

4.982

1750331

LINEARITY-125%

Metformin

4.978

2621620

LINEARITY-250%

Metformin

4.977

3501905

LINEARITY-375%

Metformin

4.975

4374337

LINEARITY-500%

Metformin

4.976

5253057

Table 8: Linearity Studies Of Gliclazide

S.No

Sampel Name

Inj

Name

RT

Area

LINEARITY-10%

Gliclazide

3.223

1544038

LINEARITY-20%

Gliclazide

3.221

2364521

LINEARITY-40%

Gliclazide

3.218

3156465

LINEARITY-60%

Gliclazide

3.213

3974279

LINEARITY-80%

Gliclazide

3.211

4720111

Table 9: Results of linearity for Metformin

S.No

%CONC

AREA

62.5

1750331

125

2621620

250

3501905

375

4374337

500

5253057

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Fig.1: Results of linearity for Metformin

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RESEARCH ARTICLE
Department of Pharmaceutical Analysis

Table 10: Results of linearity for Gliclazide

SNO

%CONC

Fig. 2: Results of linearity for Gliclazide

AREA

10

1544038

25

2364521

40

3156465

65

3974279

80

4720111

Table 11: Specificity

S.No

Peak name

Retention time

Diluent

No peaks are observed at retention time of main

peak

Placebo

No peaks are observed at the retention time of

main peak

3
4

Main peak (Metformin)

Main peak (Gliclazide )

Gliclazide - 5.001
Metformin - 3.232

Table 12: Limit of Detection

S.NO

DRUG

LOD(ppm)

S/n

Metformin

6.134

244.524

Gliclazide

5.474

274.027

RESULTS OF LOD FOR METFORMIN AND GLICLAZIDE

LIMIT OF QUANTITATION:

Lowest amount of analyte in a sample that can be quantitated with suitable accuracy

and

precision. It was calculated from signal to noise ratio.

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RESEARCH ARTICLE
Department of Pharmaceutical Analysis

Table 13: Limit of Quantitation

S.NO

DRUG

LOQ(ppm)

S/n

1
2

Metformin
Gliclazide

20.50
18.25

244.524
274.027

Table 14: Robustness for Metformin Gliclazide

S.No Sample name

Inj Name

Rt

Area

Std 2(flow1)

Metformin

6.165

Std 2(flow2)

Metformin

Std 2(Temp1)

Std 2(Temp2)

plate
count

Resolution

Tailing

4351433

8.307

1.243

7701

4.154

2883646

7.537

1.192

6211

Metformin

6.130

4351835

8.339

1.210

7487

Metformin

4.150

2874923

7.577

1.190

5954

Table 15: Robustness for Gliclazide

S.No

Sample name

Inj

Name

Rt

Area

Tailing

plate
count

Std 2(flow1)

Gliclazide

3.992

3941463

1.125

5203

Std 2(flow2)

Gliclazide

2.688

2582834

1.109

4342

Std 2(Temp1)

Gliclazide

3.962

3923126

1.136

5106

Std 2(Temp2)

Gliclazide

2.686

2592639

1.100

4348

SUMMARY AND CONCLUSION

Development of new analytical methods for the determination of drugs in pharmaceutical dosage
forms is more important in pharmacokinetic, toxicological and biological studies. Today
pharmaceutical analysis entails much more than the analysis of active pharmaceutical ingredients
or the formulated product. The pharmaceutical industry is under increased scrutiny from the
government and the public interested groups to contain costs and at consistently deliver to market
safe, efficacious product that fulfill unmet medical needs. The pharmaceutical analyst plays a
major rule in assuring identity, safety, efficacy, purity, and quality of a drug product. The need for
pharmaceutical analysis is driven largely by regulatory requirements. The commonly used tests of
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RESEARCH ARTICLE
Department of Pharmaceutical Analysis

pharmaceutical analysis generally entail compendia testing method development, setting

specifications, and method validation. Analytical testing is one of the more interesting ways for
scientists to take part in quality process by providing actual data on the identity, content and purity
of the drug products. New methods are now being development with a great deal of consideration
to worldwide harmonization. As a result, new products can be assured to have comparable quality
and can be brought to international markets faster [11]. Pharmaceutical analysis occupies a pivotal
role in statuary certification of drugs and their formulations either by the industry or by the
regulatory authorities. In industry, the quality assurance and quality control departments play
major role in bringing out a safe and effective drug or dosage form. The current good
manufacturing practices (CGMP) and the Food Drug Administration (FDA) guidelines insist for
adoption of sound methods of analysis with greater sensitivity and reproducibility. Therefore, the
complexity of problems encountered in pharmaceutical analysis with the importance of achieving
the selectivity, speed, low cost, simplicity, sensitivity, specificity, precision and accuracy in
estimation of drugs [12].
Table 16: Summary Report of HPLC validation
Validation Parameters

Acceptance Criteria

HPLC Results

Precision

The %RSD of peaks

Metformin

Gliclazide

obtained from the 6

0.08

0.10

The % recovery at each

Metformin

Gliclazide

level shall be NLT

100

99

The Correlation

Metformin

Gliclazide

coefficient shall be NLT

0.99

0.99

replicate injections
should be NMT 2.0%
Accuracy

98.0% and NMT 102.0%

of the added amount.
Linearity

0.999
Robustness

All the system suitability

The system suitability parameters passed

for all the Conditions.

parameters should pass

for all the conditions.

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RESEARCH ARTICLE
Department of Pharmaceutical Analysis

For 6 replicate injections

System Suitability

Metformin

The %RSD NMT 2.0%

0.6

Gliclazide
0.7

Tailing factor NMT 2.0%

1.213

1.105

Plate Count NLT 3000

6782

4670

REFERENCES
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method development and validation for the estimation of Gliclazide in bulk and
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5. T.maeda, T.yamaguchi and M Hashimoto, J. Chromatogr. B Biomed.sci appl., 1981,223,357363.
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7. http://wiki.medpedia.com/Clinical:Actoplus_met (pioglitazone_hydrochloride_and_
metformin_hydrochloride)
8. http://jpronline.info/article/view/628
9. http://en.wikipedia.org/wiki/Pioglitazone
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08/metformin-and-pioglitazone-hplc-estimation.
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